Figure 4.

Spillover Affects IMC Data and Can Be Corrected Using Our Compensation Strategy

(A) Binning the signals of an imaged ¹⁶⁶Er metal spot (to the 95th percentile of the ¹⁶⁶Er pixel values) into 20 bins with equal pixel numbers shows a linear relationship between ¹⁶⁶Er and ¹⁶⁷Er over several orders of magnitude (upper panel). The relationship between ¹⁶⁶Er and ¹⁶⁸Er appears linear but saturates at the higher counts (lower panel).

(B) Representative image section of a breast cancer tissue sample imaged by IMC. Top row shows uncompensated images of ¹⁶⁶Er (used to label antibody to carbonic anhydrase), ¹⁶⁷Er (no antibody labeled with this metal), and ¹⁶⁸Er (used to label anti-KI67). The bottom row shows corresponding NNLS compensated images. For visualization, a 3 × 3 pixel median filter was used to reduce noise. Scale bars, 20 μm. Red arrows indicate part of the image where low signal was removed by compensation.

(C) Segmentation mask shown on representative images described in (B). The mean pixel intensities of the signals observed in the indicated channels per cell are displayed.

(D) Scatterplots from single-cell segmentation data from the IMC images before (left) and after (right) compensation. Arcsinh transformed ion counts (cofactor of 2) are shown.